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Aim To perform with 100% accuracy the DAT procedure and elution procedure with correct
interpretation of results.
Introduction DAT
The direct antiglobulin test (DAT) is used to detect in vivo red blood cell sensitization.
Washed red blood cells from the patient are directly tested with antiglobulin serum. The
specimen of choice for the DAT is an EDTA specimen. If a clotted specimen is used a
false positive result may be obtained due to non-specific binding of complement to the red
blood cells in-vitro. EDTA binds calcium which is necessary for complement activation.
The focus of this lab is to determine sensitization of baby’s red blood cells, but the DAT
is a procedure which may also be performed on other types of patients as well as donor
units.
The DAT is utilized in the blood bank to aid in the diagnosis of a variety of
conditions such as:
If a patient has a positive DAT, an elution procedure should be performed. A critical part
of the elution procedure includes the patient history. The patient's diagnosis, transfusion
and pregnancy history, and recent/current medications are vital pieces of informa-
tion in working up a positive DAT.
Elution
The next two (2) laboratories are blood bank tests used in the evaluation of hemolytic
disease of the newborn (HDN). Immune antibodies are stimulated as a result of exposure
to red blood cell antigens which an individual does not possess. If a woman has been
exposed to foreign red cell antigens (most commonly as a result of transfusion or previous
pregnancy), she may form immune (IgG) antibodies capable of crossing the placenta and
attaching to antigen positive fetal cells. This causes the baby's red blood cells to be coated
with antibody and destroyed. The resulting effects on the child are collectively termed
hemolytic disease of the newborn (HDN). The severity of the HDN varies and is
dependent upon the antibody involved. The most severe HDN is due to D negative
mothers who have formed immune anti-D that crosses the placenta and destroys the D
positive cells of her D positive fetus. If the HDN is severe, the baby will need an
exchange transfusion with blood compatible with the baby and the mother.) The
mildest and most common form of HDN is due to maternal ABO antibodies. This is
detected in group A or B infants of group O mothers. Phototherapy is the treatment of
choice for ABO HDN. Antibodies to “other” blood group antigens may cause varying
degrees of HDN.
Principle Part 1 of this lab is to determine whether an infant's red blood cells have been sensitized
by maternal antibody by performing the direct antiglobulin test (DAT). Please remember
that the DAT is a general procedure that is performed on babies, adults and donor
units. The purpose of the test in this laboratory session is to detect sensitization of baby
cells by maternal antibody, but this test is also applicable in many other situations.
The DAT procedure detects in-vivo sensitization of red blood cells. If a negative result
is obtained, no sensitization has occurred. A positive result indicates in-vivo sensitization
has occurred and further testing is required.
The specimen for testing is obtained from the baby's umbilical cord immediately following
birth. A syringe is used to withdraw a specimen to prevent contamination with Wharton's
jelly. Wharton's jelly is a gelatinous substance which coats the umbilical cord. If the cord
When a positive cord blood DAT is obtained a series of steps are required to identify the
offending antibody. The first step in determining the identity of the coating antibody is
to perform a Type and Screen on the mom and a ABO/D typing on the infant. Please
remember that a reverse type cannot be performed on babies, as the ABO antibodies
present in their serum/plasma is of maternal origin. The results of the antibody screen
on the mother will determine which type of elution to perform, as some elution procedures
are best at recovering alloantibodies, while others are best at recovering immune ABO
antibodies. If the mother's antibody screen is positive, a panel study is done on the
mother's serum. An acid elution must be performed on the infant's cord cells to verify that
the antibody in the mother's serum is, indeed, coating the baby's cells. If the mom's
screens are negative and she is ABO incompatible with her infant then a heat elution is
performed on the infant's cord cells to check for an ABO antibody.
Part 2 of this lab will involve the performance of an elution on the infant's cord cells to
determine the specificity of the maternal antibody coating the infant's red blood cells. In
an elution procedure, the cells are washed free of all unbound antibody and a procedure
is performed on the cells to cause the release of the antibody molecules from the cells into
a solution. The eluate is then tested against reagent red cells to determine the specificity.
Remember that any antibody recovered in the eluate prepared from the infant's cells is of
maternal origin.
Please keep the following flow chart handy as you perform the cordblood DAT
testing.
DAT results
NEGATIVE POSITIVE
Rh on cord cells
DAT 1. Label a tube for each cord specimen using the mother's full name and hospital
number.
Procedure
While the type and screen on the mother is incubating, perform an ABO/D on
the baby's cells. A reverse type cannot be performed on an infant since all
antibodies in the baby are of maternal origin. After all results are in, look at
the flow chart to determine which eluate will be performed next week.
The red blood cells for any elution technique must be thoroughly washed to remove all
Heat Elution
antibody except that bound to the cells. Six to eight washes with large volumes of
Introduction
saline are usually sufficient. *Adequacy of washing is determined by testing saline
from the last wash against reagent screen cells for the presence of antibody. If positive
reactions are obtained with the last wash this is an indication that the red blood
The heat elution technique is used to remove antibodies from red blood cells coated
in-vivo. It is used primarily to recover and identify ABO antibodies in cases of
hemolytic disease of the newborn (HDN) and in patients who have been transfused with
non-ABO-identical blood or blood components. The coated red blood cells are
thoroughly washed, an albumin solution is added and the cells are heated to 56 C. This
amount of heat will cause disruption of the antigen-antibody bond, the antibody will
“pop off” the coated rbcs into the albumin solution. After sufficient heating the
solution is centrifuged and the supernatant fluid (eluate) is removed. The supernatant
will be very hemolyzed and you may need a lamp to provide a lighted background to
view the eluate/rbc interface. Contaminating the eluate with rbcs may cause false
positive reactions in the test. The supernatant fluid will contain the antibodies that were
coating the cells. The eluate is tested against reagent red blood cells to determine the
specificity.
1. See page 1
Reagents 2. 6% bovine albumin, prepared by diluting 22% or 30% bovine albumin with saline
3. Cord blood red blood cells from previous lab
4. 56°C water bath
Testing the 1. Label 10, 12 x 75 test tubes (eluate (EL) = A1, B, S1, S2, and S3; last wash (LW) =
A1, B, S1, S2, and S3) and patient initials.
Eluate
2. Add one (1) drop of the reagent red cells to each of the properly labeled tubes.
3. To the five (5) tubes labeled “EL”, add three (3) to four (4) drops of the eluate.
Interpretation 1. If “last wash” tubes are negative, any agglutination of reagent cells with
eluate indicates recovery of antibodies from the original cells.
2. If the last wash is positive with any of the test cells, the original
cell sample was probably under washed and the elution
If the mother is group O and the baby is group A or B, it is not unusual to obtain
positive reactions with both the A and B cells. This reactivity is due to the anti-A,B
present in the group O mother’s serum which has coated the baby’s rbcs. This antibody
will react with both A and B cells.
Use the interpretation chart above along with your common sense to interpret your
data. If the mom is group O and the baby is group A, it is impossible to elute an
immune anti-B from the baby cellls. Think your interpretation through carefully.
For the example below the mom is group O, antibody screen negative and the baby is
group A with a positive DAT. The following elution results were obtained.
A1 cells B cells S1 S2 S3
Eluate 4+ 2+ 0 0 0
Last Wash Control 0 0 0 0 0
Date_________________________________
Cordblood DAT
Recording Results
ABO/Rh
Interp
Duu
DCC
D
B
A,B
DAT
Date_________________________________
Elution Studies
Recording Results
Mother's Name
Hospital Number
Baby's DAT
A1
S1
S2
S3
2. List four (4) clinical conditions for which the DAT is useful. (2 points)
4. What is the source of the specimen used for the DAT on newborns?
6. List the three types of HDN from mildest to most severe and state the antibody specificity involved in
each. (3 points)
8. State the additional testing which must be performed when an infant has a positive DAT. Be sure to
state which test is performed on mom and which on baby. (1.5 points)
9. What must be done for the infant if the HDN is severe at birth? (1 point)
2. List four (4) pieces of information that are useful in the workup of a positive DAT. (2 points)
3. What determines which type of elution procedure to use for an HDN workup? (1 point)
5. In the elution procedure, why must the cells be thoroughly washed? (1 point)
6. What is the most probable cause of positive results with the last wash control? (1 point)
8. What does lack of agglutination of the test cells by the eluate indicate? (0.5)
Baby is A positive
Mom is 0 positive, screens are negative
A1 cells B cells S1 S2 S3
Eluate 4+ s 2+ 0 0 0
Last Wash 0 0 0 0 0
Interpretation:
10. When the mother is group O and the baby is A or B why is it common to get reactions in the eluate with
both the A and B cells? (1 point)